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Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates.

ABSTRACT: Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential.

SUBMITTER: Nouws S

PROVIDER: S-EPMC7474065 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Shiga toxin-producing Escherichia coli (STEC) are considered to be a significant threat to public health due to the severity of gastrointestinal symptoms associated with human infection. In England STEC O157 is the most commonly detected STEC serogroup, however, the implementation of PCR at local hospital laboratories has resulted in an increase in the detection of non-O157 STEC. The aim of this study was to evaluate the use of whole genome sequencing (WGS) for routine public health surveillance of non-O157 STEC by comparing this approach to phenotypic serotyping and PCR for subtyping the stx-encoding genes. Of the 102 isolates where phenotypic and genotypic serotyping could be compared, 98 gave fully concordant results. The most common non-O157 STEC serogroups detected were O146 (22) and O26 (18). All but one of the 38 isolates that could not be phenotypically serotyped (designated O unidentifiable or O rough) were serotyped using the WGS data. Of the 73 isolates where a flagella type was available by traditional phenotypic typing, all results matched the H-type derived from the WGS data. Of the 140 sequenced non-O157 isolates, 52 (37.1%) harboured stx1 only, 42 (30.0%) had stx2 only, 46 (32.9%) carried stx1 and stx2. Of these, stx subtyping PCR results were available for 131 isolates and 121 of these had concordant results with the stx subtype derived from the WGS data. Of the 10 discordant results, non-specific primer binding during PCR amplification, due to the similarity of the stx2 subtype gene sequences was the most likely cause. The results of this study showed WGS provided a reliable and robust one-step process for characterization of STEC. Deriving the full serotype from WGS data in real time has enabled us to report a higher level of strain discrimination while stx subtyping provides data on the pathogenic potential of each isolate, enabling us to predict clinical outcome of each case and to monitor the emergence of hyper-virulent strains.

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Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates.

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